Throughout this application, various references are referred to in parentheses. Disclosures of these publication in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.
Microfibrils, 10-14 nm in diameter, are extracellular matrix proteins which play important roles in the assembly and maintenance of elastic fibers.1.2 A bovine microfibril-associated glycoprotein (MAGP) with MW 31 kDa was discovered in 1986,3,4 and then cloned in 1994.5 Kobayashi et al. Reported a bovine MAGP-36 with calcium-binding and fibrinogen-like domains, with a tissue distribution uniquely limited to aorta.6 A human MAGP, implicated to Smith Magenis syndrome, was sequenced in 1995.7 
Immunoglobulin (IgG) purified from the aortic wall of patients with abdominal aortic aneurysms (AAAs) is immunoreactive with a human aortic protein that is homologous to MAGP-36.8.8 As described herein, the protein has been purified and partially sequenced. It has vitronectin and fibrinogen-like domains, along with a putative calcium-binding domain.9 It is designated Aortic Aneurysm-Autoantigenic Proteinxe2x80x9440 kDa or Aortic-Aneurysm-Associated Protein-40 (AAAP-40).9 
Another matrix protein detected in human embryonic tissue (sulfated protein 30 kDa=SP-30) has been reported to be immunoreactive with monoclonal antibodies against human vitronectin, and it co-distributes in tissue with the Protein that is immunoreactive with antibody against MAGP-31. A sequence in AAAP-40 matches residues #230-240 in human vitronectin. It was demonstrated that AAAP-40 was immunoreactive with rabbit anti-human vitronectin antibody. In addition, the experiments described in this application were carried out to clone the cDNA encoding AAAP-40, express it as a recombinant, and assign it conclusively as a human MAGP.
Nomenclature of the microfibrillar proteins associated with the elastin fiber is confusing. A principal component of the microfibril is fibrillin (fib-15), discovered by Sakai, et al.,1 and Marfan""s syndrome has been traced to mutations in the gene for fibrillin on chromosome 15.2 3 4 A bovine microfibrillar protein (Mr apx. 31 kDa) was discovered in 1986 by Gibson, et al.,5 6 who coined the term xe2x80x9cmicrofibril-associated glycoproteinxe2x80x9d(MAGP). Bashir, et al. have also cloned the gene for this protein.7 Kobayashi, et al. reported a 36 kDa calcium-binding protein, also in cow, with tissue distribution uniquely limited to the aorta (MAGP-36).8 The second human MAGP (deduced MW 21 kDa) was recently reported to have an open reading frame of 255 amino acids and to be linked to Smith Magenis syndrome.9 The authors of the paper describing the Smith Magenis protein prefer the abbreviation xe2x80x9cMFAPxe2x80x9d, to avoid confusion with abbreviations for microfilamentous proteins.
It has been reported that IgG from the aortic wall of patients with abdominal aortic aneurysms (AAA) is immunoreactive with a human aortic protein (MW apx. 80 kDa) that has features of the bovine aortic protein of Kobayshi, et al. (MAGP-36).10 MAGP-36 occurs in nature as a disulfide-bonded dimer, so further tissue extractions under reducing conditions as described by Prosser, et al. were carried out as described herein.11 This approach has led to the partial characterization of a protein of apx. 40 kDa that is immunoreactive with AAA IgG. This protein is called Aortic Aneurysm-Autoantigenic Proteinxe2x80x9440 kDa or Aortic-Aneurysm-Associated Protein-40 (AAAP-40). The present application describes its partial sequence and suggests that, since it is the third human microfibrillar protein to be described, it be called MAGP-3.
This invention provides an isolated protein of approximately 40 kDa which is purified from human aortic tissue and immunoreactive with AAA-associated immunoglobulin. This invention provides an isolated protein has the amino acid sequence set forth in SEQ ID NO:1.
This invention provides a recombinantly produced human aortic protein which is immunoreactive with both AAA-associated immunoglobulin and human kappa immunoglobulin and having a molecular weight of approximately 24 kDa-28 kDa.
This invention further provides an isolated nucleic acid molecule encoding a human aortic protein. In an embodiment, the isolated nucleic acid, cDNA, designated AAAP-CL1, encodes a human aortic protein which is immunoreactive with both AAA-associated immunoglobulin and human kappa immunoglobulin and has a molecular weight of approximately 24 kDa-28 kDa. In another embodiment, the isolated cDNA, designated AAAP-CL5, encodes a human aortic protein which is immunoreactive with both AAA-associated immunoglobulin and human kappa immunoglobulin and has a molecular weight of approximately 24 kDa-28 kDa. In yet another embodiment, the isolated cDNA, designated AAAP-CL4, encodes a human aortic protein which is immunoreactive with both AAA-associated immunoglobulin and human kappa immunoglobulin and has a molecular weight of approximately 24 kDa-28 kDa.
This invention provides a recombinantly produced human aortic protein which is immunoreactive with AAA-associated immunoglobulin. In an embodiment, the protein is of a molecular weight of approximately 40 kDa.
This invention provides a purified recombinant human aortic protein of approximately 24 kDa-28 kDa having the amino acid sequence set forth in SEQ ID NO:2 which is immunoreactive with both AAA-associated immunoglobulin and human kappa immunoglobulin.
This invention provides a purified recombinant human aortic protein of approximately 28 kDa having the amino acid sequence set forth in SEQ ID NO:3 which is immunoreactive with both AAA-associated immunoglobulin and human kappa immunoglobulin.
This invention provides a purified recombinant human aortic protein of approximately 24 kDa-28 kDa having the amino acid sequence set forth in SEQ ID NO:4 which is immunoreactive with both AAA-associated immunoglobulin and human kappa immunoglobulin.
This invention provides an antibody directed to a purified human protein. This invention further provides an antibody capable of specifically recognizing human aortic protein.
This invention provides a method of diagnosing AAA disease in a subject which comprises: (a) obtaining a suitable sample from the subject; (b) contacting the sample with the protein of either the isolated protein of approximately 40 kDa which is purified from human aortic tissue and immunoreactive with AAA-associated immunoglobulin or the isolated protein having the amino acid sequence set forth in SEQ ID NO:1 under conditions permitting the protein to bind to AAA-associated immunoglobulin if present in the sample; and (c)determining the presence of bound protein, the presence of bound protein being indicative of AAA disease, thereby diagnosing AAA disease in the subject.
This invention provides a pharmaceutical composition for alleviating AAA disease in a subject which comprises the protein of approximately 40 kDa which is purified from human aortic tissue and immunoreactive with AAA-associated immunoglobulin and a pharmaceutically acceptable carrier.
This invention provides a method of alleviating AAA disease in a subject which comprises administering to the subject an amount of the aforementioned composition effective to induce tolerance to antigenic AAA protein in the subject.
This invention provides a method of diagnosing AAA disease in a subject which comprises: (a) obtaining a suitable sample from the subject; (b) contacting the sample with the protein selected from any of the following proteins: the isolated protein of approximately 40 kDa which is purified from human aortic tissue and immunoreactive with AAA-associated immunoglobulin; the isolated protein of approximately 40 kDa which is purified from human aortic tissue and immunoreactive with AAA-associated immunoglobulin having the amino acid sequence set forth in SEQ ID NO:1; a recombinantly produced human aortic protein which is immunoreactive with AAA-associated immunoglobulin; a recombinantly produced human aortic protein which is immunoreactive with AAA-associated immunoglobulin and is of a molecular weight of approximately 40 kDa; a purified recombinant human aortic protein of approximately 24 kDa-28 kDa having the amino acid sequence set forth in SEQ ID NO:2 which is immunoreactive with both AAA-associated immunoglobulin and human kappa immunoglobulin; a purified recombinant human aortic protein of approximately 28 kDa having the amino acid sequence set forth in SEQ ID NO:3 which is immunoreactive with both AAA-associated immunoglobulin and human kappa immunoglobulin; a purified recombinant human aortic protein of approximately 24 kDa-28 kDa having the amino acid sequence set forth in SEQ ID NO:4 which is immunoreactive with both AAA-associated immunoglobulin and human kappa immunoglobulin under conditions permitting the protein to bind to AAA-associated immunoglobulin if present in the sample; and (c) determining the presence of bound protein, the presence of bound protein being indicative of AAA disease, thereby diagnosing AAA disease in the subject. In an embodiment, the subject may be a mammal, for example a human subject.
This invention provides a pharmaceutical composition for alleviating AAA disease in a subject which comprises the protein is selected from any of the following proteins: the isolated protein of approximately 40 kDa which is purified from human aortic tissue and immunoreactive with AAA-associated immuinoglobulin; the isolated protein of approximately 40 kDa which is purified from human aortic tissue and immunoreactive with AAA-associated immunoglobulin having the amino acid sequence set forth in SEQ ID NO:1; a recombinantly produced human aortic protein which is immunoreactive with AAA-associated immunoglobulin; a recombinantly produced human aortic protein which is immunoreactive with AAA-associated immunoglobulin and is of a molecular weight of approximately 40 kDa; a purified recombinant human aortic protein of approximately 24 kDa-28 kDa having the amino acid sequence set forth in SEQ ID NO:2 which is immunoreactive with both AAA-associated immunoglobulin and human kappa immunoglobulin; a purified recombinant human aortic protein of approximately 28 kDa having the amino acid sequence set forth in SEQ ID NO:3 which is immunoreactive with both AAA-associated immunoglobulin and human kappa immunoglobulin; a purified recombinant human aortic protein of approximately 24 kDa-28 kDa having the amino acid sequence set forth in SEQ ID NO:4 which is immunoreactive with both AAA-associated immunoglobulin and human kappa immunoglobulin and a pharmaceutically acceptable carrier.
This invention provides a kit for detecting the presence of AAA-associated immunoglobulin in a sample, which comprises: (a) a solid support having a plurality of covalently linked probes which may be the same or different, each probe of which comprises a human aortic protein which is capable of binding AAA-associated immunoglobulin; and (b) a means for determining the presence of AAA-associated immunoglobulin bound to the human aortic protein. In an embodiment of the kit, the human aortic protein is selected from any of the following proteins: the isolated protein of approximately 40 kDa which is purified from human aortic tissue and immunoreactive with AAA-associated immunoglobulin; the isolated protein of approximately 40 kDa which is purified from human aortic tissue and immunoreactive with AAA-associated immunoglobulin having the amino acid sequence set forth in SEQ ID NO:1; a recombinantly produced human aortic protein which is immunoreactive with AAA-associated immunoglobulin; a recombinantly produced human aortic protein which is immunoreactive with AAA-associated immunoglobulin and is of a molecular weight of approximately 40 kDa; a purified recombinant human aortic protein of approximately 24 kDa-28 kDa having the amino acid sequence set forth in SEQ ID NO:2 which is immunoreactive with both AAA-associated immunoglobulin and human kappa immunoglobulin; a purified recombinant human aortic protein of approximately 28 kDa having the amino acid sequence set forth in SEQ ID NO:3 which is immunoreactive with both AAA-associated immunoglobulin and human kappa immunoglobulin; a purified recombinant human aortic protein of approximately 24 kDa-28 kDa having the amino acid sequence set forth in SEQ ID NO:4 which is immunoreactive with both AAA-associated immunoglobulin and human kappa immunoglobulin.
This invention provides a kit for detecting the presence of an elevated or abnormal level of human aortic protein in a sample, which comprises:(a) a plurality of covalently linked probes which may be the same or different, each probe of which comprises any one of a AAA-associated immunoglobulin and a human kappa immunoglobulin which is capable of binding human aortic protein; and (b) a means for determining the elevated or abnormal level of human aortic protein bound to AAA-associated immunoglobulin or human kappa immunoglcbulin by comparison to a normal level of human aortic protein.